In view of the importance of inorganic phosphate and related reactions to cellular metabolism, we are planning a detailed investigation into the molecular mechanism of inorganic phosphate transport across the inner mitochrondrial membrane and how it is modified in senescence. Taking advantage of our identification, purification and reconstitution of the mitochrondrial phosphate transport protein from beef heart, rat heart, and insect flight muscle, our pilot studies have demonstrated a significant decline in the phosphate transport protein concentration and in the energy-linked calcium transport activity of mitochrondria with senescence. We are planning to use the phosphate transporter incorporated into liposomes and in its native mitochrondrial membrane to study the kinetics of the transport in detail and whether it is electrophoretic or electroneutral at all pH's. We will carry out experiments designed to establish the presence of a protein proton binding site separate from or part of the phosphate binding site. We will characterize the protein's topographic orientation in the membrane and expect that an identification of peptides or amino acids involved in phosphate and proton binding will permit us to characterize the "movement" of the phosphate/proton binding site across the membrane. We will attempt to purify the ruthenium red sensitive mitochrondrial calcium transport protein and expect that these studies will help us understand the senescence related changes in transporter activities of senescent mitochondria and cell membranes in general.